What I did:
The final project was worked on as well as virtual screening.
Analysis:
Control ligands were found although when prepping them something went wrong because the run for screening kept giving me a failure- "fatal error" response. This could be because the ligands might have been prepped wrong or the ligand that was found bound in the Human PSP wasnt a decent ligand to gather data from in the first place. Although, the human PSP containing a ligand was aligned with the Vc PSP in order to find the Vc PSPs active site. This can happen due to the low RMS value received from the alignment.
Image 1: PyMol representation of Vc PSP (3n28) colored purple, aligned over the green colored human PSP (1l7p) to show the active site location of the target protein VcPSP.
12042014- No pictures or analysis of lab procedures. I added some-- (John G, 12/12/14)
Weeks 11,12,13:
The pellets were then lysed and sonicated, after the sonication we stored the protein (without syringe filtering) for 3 days (a weekend). When we came back the next week we proceeded with purification. The MidiPrep went well, except for a failure in labeling the 0-6 samples. The next step would have been FPLC, but during the past due syringe filtering the protein was spilled and the project terminated. The next step is to move back to Vc PSP and begin virtual which will be weeks 14, and 15.
Weeks 8,9,10:
Image Weeks8,9,10: Pymol representation of the protein RpFabG, shown as multicolor cartoon. Analysis: The protein was expressed and then after spinning down, the protein semi solidified in the storage tube. This could be that RpFABG becomes unstable after 24 hours without being purified first. Next time after spinning down, immediate purification must occur.
Worked on a new target, RpFABG for mass production of the protein for Oscar V.: Xenia and I prepared 4L total of LB for the expression. But we first made a small overnight culture using Oscar V's master plate, then added 10 ml of the overnight culture to each of the 1L flasks (4 in total). We then brought the OD600 to approx. 0.315 and induced the expression with IPTG at room temp for 16 hrs. Once the 16 hours was up, we spun down the bacteria and collected the pellet for storage. 1162014- Where is week 8,9,10?
Weeks 5, 6, & 7:
9232014- Cant view the PCR image, and you can add more detail to your captions Figure 3 Week 7: Primary PCR results trial one; lane two is 1kb ladder, lane 4 is John G oligo mix (not seen), lane 6 is Keenan Wu oligo mix (seen as smear). Figure 2 Week 6: Tail primer design results for vibrio cholerae Phosphoserine phosphotase. Figure 1 Week 5: Pymol refresher image, showing the protein being investigated in the protocol.
Weeks 5, 6, and 7 Analysis:
In week 5 pymol refresher was done with the purpose of getting pymol skills up to date for research this semester. Week 6 was devoted to designing the tail primers and ordering oligos for the target protein Phosphoserine Phosphotase from Vibrio cholerae. Week 7, primary PCR failed and is being redone with a longer annealing time. This "fail" could be a result of a bad oligo mix, or not. This will be checked by both moving on to secondary PCR and redoing Primary PCR.
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9302014- Good Job, Keep up the good work.
Weeks 3&4:
Figure 2 Week 4: Final results for designing Oligos via DNA Outworks.
Figure 1 Week 3: Gel electro-phoresis of protein pgBR22 Plasmid coding sequence using M13 Rev primers. Lane 1:empty, Lane 2:100bp ladder, Lane 3 :Sol'n A (1:10000), Lane 4 : Sol'n B (1:1000), Lane 5 :Sol'C (1:100), Lane 6 :Sol'n D (the uncut pgBR22).
Weeks 3&4 Analysis: The Gel worked out as hypothesized, there are three subsequent bands following the ladder in the appropriate lane. Although each is supposed to get darker due to increasing concentration, this gel is not the case. This is an indication that a mix up occurred in the sequence of solutions when being placed in the wells. This shows that the protein was taken up by the E. coli. ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
982014- Remember to include an analysis of your bacterial cultures
Weeks 1&2:
Figure 2 Week 2: No growth seen by Bacterial culture of E.coli DH5alpha with plasmid DNA pNIC-Bsa4, one containing a10ul sample of our E.coli with inserted DNA (right) and 50ul sample (left).
Figure 1 Week 1: Nano-drop results on pGBR-22 sample.
Analysis of Weeks 1&2:
In figure 1 you can see that the DNA absorbs the most light at wavelength of 260, this indicates the maximum absorbency of light wavelength by pGBR-22. In Figure 2 you can see that the bacteria did not grow, this may be in account for human error.
Weeks 14,15:
What I did:The final project was worked on as well as virtual screening.
Analysis:
Control ligands were found although when prepping them something went wrong because the run for screening kept giving me a failure- "fatal error" response. This could be because the ligands might have been prepped wrong or the ligand that was found bound in the Human PSP wasnt a decent ligand to gather data from in the first place. Although, the human PSP containing a ligand was aligned with the Vc PSP in order to find the Vc PSPs active site. This can happen due to the low RMS value received from the alignment.
Image 1: PyMol representation of Vc PSP (3n28) colored purple, aligned over the green colored human PSP (1l7p) to show the active site location of the target protein VcPSP.
12042014- No pictures or analysis of lab procedures. I added some-- (John G, 12/12/14)
Weeks 11,12,13:
The pellets were then lysed and sonicated, after the sonication we stored the protein (without syringe filtering) for 3 days (a weekend). When we came back the next week we proceeded with purification. The MidiPrep went well, except for a failure in labeling the 0-6 samples. The next step would have been FPLC, but during the past due syringe filtering the protein was spilled and the project terminated. The next step is to move back to Vc PSP and begin virtual which will be weeks 14, and 15.Weeks 8,9,10:
Image Weeks 8,9,10: Pymol representation of the protein RpFabG, shown as multicolor cartoon.
Analysis: The protein was expressed and then after spinning down, the protein semi solidified in the storage tube. This could be that RpFABG becomes unstable after 24 hours without being purified first. Next time after spinning down, immediate purification must occur.
Worked on a new target, RpFABG for mass production of the protein for Oscar V.: Xenia and I prepared 4L total of LB for the expression. But we first made a small overnight culture using Oscar V's master plate, then added 10 ml of the overnight culture to each of the 1L flasks (4 in total). We then brought the OD600 to approx. 0.315 and induced the expression with IPTG at room temp for 16 hrs. Once the 16 hours was up, we spun down the bacteria and collected the pellet for storage.
1162014- Where is week 8,9,10?
Weeks 5, 6, & 7:
9232014- Cant view the PCR image, and you can add more detail to your captionsFigure 3 Week 7: Primary PCR results trial one; lane two is 1kb ladder, lane 4 is John G oligo mix (not seen), lane 6
is Keenan Wu oligo mix (seen as smear).
Figure 2 Week 6: Tail primer design results for vibrio cholerae Phosphoserine phosphotase.
Figure 1 Week 5: Pymol refresher image, showing the protein being investigated in the protocol.
Weeks 5, 6, and 7 Analysis:
In week 5 pymol refresher was done with the purpose of getting pymol skills up to date for research this semester. Week 6 was devoted to designing the tail primers and ordering oligos for the target protein Phosphoserine Phosphotase from Vibrio cholerae. Week 7, primary PCR failed and is being redone with a longer annealing time. This "fail" could be a result of a bad oligo mix, or not. This will be checked by both moving on to secondary PCR and redoing Primary PCR.
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
9302014- Good Job, Keep up the good work.
Weeks 3&4:
Figure 2 Week 4: Final results for designing Oligos via DNA Outworks.
Figure 1 Week 3: Gel electro-phoresis of protein pgBR22 Plasmid coding sequence using M13 Rev primers. Lane 1:empty, Lane 2:100bp ladder, Lane 3 :Sol'n A (1:10000), Lane 4 : Sol'n B (1:1000), Lane 5 :Sol'C (1:100), Lane 6 :Sol'n D (the uncut pgBR22).Weeks 3&4 Analysis: The Gel worked out as hypothesized, there are three subsequent bands following the ladder in the appropriate lane. Although each is supposed to get darker due to increasing concentration, this gel is not the case. This is an indication that a mix up occurred in the sequence of solutions when being placed in the wells. This shows that the protein was taken up by the E. coli.
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
982014- Remember to include an analysis of your bacterial cultures
Weeks 1&2:
Figure 2 Week 2: No growth seen by Bacterial culture of E.coli DH5alpha with plasmid DNA pNIC-Bsa4,
one containing a10ul sample of our E.coli with inserted DNA (right) and 50ul sample (left).
Figure 1 Week 1: Nano-drop results on pGBR-22 sample.
Analysis of Weeks 1&2:
In figure 1 you can see that the DNA absorbs the most light at wavelength of 260, this indicates the maximum absorbency of light wavelength by pGBR-22. In Figure 2 you can see that the bacteria did not grow, this may be in account for human error.